Technical Reference #1682

1682.

Exocytosis of Post-Golgi Vesicles is Regulated by Components of the Endocytic Machinery Jyoti K. Jaiswal, Victor M. Rivera,2 and Sanford M. Simon, The Rockefeller University, ARIAD Gene Therapeutics, Inc, Cell, 137(1682), 1317, (2009)
Link To Paper

Abstract:
Post-Golgi vesicles target and deliver most biosynthetic cargoes to the cell surface. However, the molecules and mechanisms involved in fusion of these vesicles are not well understood. We have employed a system to simultaneously monitor release of luminal and membrane biosynthetic cargoes from individual post-Golgi vesicles. Exocytosis of these vesicles is not calcium triggered. Release of luminal cargo can be accompanied by complete, partial, or no release of membrane cargo. Partial and no release of membrane cargo of a fusing vesicle are fates associated with kiss-and-run exocytosis, and we find that these are the predominantmodeof post-Golgi vesicle exocytosis. Partial cargo release by post-Golgi vesicles occurs because of premature closure of the fusion pore and is modulated by the activity of clathrin, actin, and dynamin. Our results demonstrate that these components of the endocytic machinery modulate the nature and extent of biosynthetic cargo delivery by post-Golgi vesicles at the cell membrane.

Materials & Methods:
Cell Culture and Treatments Human fibrosarcoma cells HT1080 were cultured inDMEMsupplemented with 10% FBS (Invitrogen, Carlsbad, CA) in 5% CO2 at 37 C. For imaging, cells were plated onto glass coverslips (Fisher Scientific, Pittsburgh, PA) or on glass bottom dishes (MatTek, Ashland, MA) and imaged in OptiMEM (Invitrogen). Cells were transfected with effectene (QIAGEN, Valencia, CA) or Lipofectamine 2000 (Invitrogen). Transiently transfected cells were imaged within 72 hr of transfection. To release the cargo from ER, an aqueous solution of AP21988 (1 mM, ARIAD Gene Therapeutics, MA) was diluted in OptiMEM and added to the cells at the final concentration of 2 mM for the desired period prior to imaging. Stocks for FM4-64, BAPTA-AM (Invitrogen) and ionomycin (Sigma Aldrich, St. Louis, MO) were prepared in DMSO and diluted appropriately in OptiMEM for use. For immunodetection, we used goat anti-dynamin II polyclonal antibody (SantaCruz Biotechnology, Santa Cruz, CA), mouse anti- CLC monoclonal antibody clone CON.1 (Covance Research Products, Denver, PA), and mouse anti-CHC monoclonal antibody clone 23 (BD Biosciences Pharmingen, San Diego, CA). For clathrin knockdown, siRNA (AACCUGCG GUCUGGAGUCAAC) described for CHC knockdown in human cells (Hinrichsen et al., 2003) and the siGLO Red transfection control siRNA were obtained from Dharmacon (Chicago, IL). Cells were cotransfected with both these RNAs using Oligofectamine (Invitrogen), and 72 to 96 hr after transfection siGLO labeling was used to identify siRNA transfected cells.

Microscopic Technique
Fluorescence Microscopy

Cell Type(s)
HT1080